Detailed Description
For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.
The specific implementation contents of the invention comprise the following parts:
first, construct BPOZ+/+、BPOZ-/-The septicemia model of the mouse analyzes the sensitivity change of the mouse to inflammation before and after BPOZ deletion, and detects the secretion levels of IL-1 beta, IL-6 and TNF-alpha in the serum of the mouse.
Second, construct BPOZ+/+、BPOZ-/-The colitis model of the mouse analyzes the sensitivity change of the mouse to inflammation before and after BPOZ deletion, including the weight of the mouse, the stool viscosity of the mouse, the hematochezia condition, the observed colon length, the ulcer area displayed by HE staining, semi-quantitative evaluation of the inflammation degree and the like.
Third, isolated culture of BPOZ+/+And BPOZ-/-In vitro analysis of primary macrophages (BMDMs) from mice was performed to analyze the secretion of IL-1. beta. by these cells under the action of classical and non-classical stimulators such as NLRP3 stimulators (ATP, MSU, CPPD, Nigericin and Poly (I: C)).
Over-expressing pro-caspase-1 and BPOZ with GFP labels in HEK293T cells, analyzing the interaction between BPOZ and caspase-1 and active subunits caspase-1p20 and caspase-1p10 thereof by a co-immunoprecipitation method, and further determining the connection mechanism of BPOZ and an inflammation signal path.
And fifthly, constructing a colitis lethal model of the mouse and analyzing the sensitivity influence of the BPOZ over-expression mouse on inflammation.
The above sequences are described below.
(I) construction of BPOZ+/+、BPOZ-/-A mouse septicemia model, analyzing the sensitivity change of the mouse to inflammation before and after BPOZ (the sequence is shown as SEQ ID NO: 1) deletion; and detecting the levels of IL-1 beta, IL-6 and TNF-alpha in the serum of the mice. The specific method and test results are as follows:
with BPOZ+/+(wild type mouse), BPOZ-/-(deletion homozygous mice) and BPOZ+/-(heterozygous mouse) mice of these three genotypes were investigated, 50mgThe dosage of the/kg is measured by injecting LPS (bacterial endotoxin) into the abdominal cavity to induce the septicemia of the mouse, and measuring the levels of IL-1 beta, IL-6 and TNF-alpha in the serum of the mouse by using an ELISA detection kit 8, 24 and 48 hours after the injection; lethality in mice was observed and calculated over 96 hours.
See (A) of FIG. 1, in BPOZ+/+、BPOZ-/-And BPOZ+/-Mice of three genotypes were the subject (n 10/group), injected intraperitoneally with LPS at a dose of 50mg/kg, and the mortality of the mice was observed and counted within 96 h. P<0.05,**p<0.001。
The results showed that BPOZ is deficient in homozygous mice (BPOZ)-/-) And heterozygous mice (BPOZ)+/-) The mortality rate of the mice is obviously higher than that of wild type mice (BPOZ)+/+) (see FIG. 1A). Wherein, BPOZ-/-And BPOZ+/+Compared with normal mice, the mice die quickly, and the death rate is obviously different. After LPS injection for 20h, no mice died in wild type mice, while BPOZ-deficient mice (BPOZ)-/-) All died, and heterozygous mice died about 50%.
See (B), (C), (D) of FIG. 1 as BPOZ+/+、BPOZ-/-And BPOZ+/-Mice of these three genotypes were the subject (n 6/group) and were treated with LPS i.p. at a dose of 50mg/kg for 3.25h followed by 50 μ L100mMATP i.p. for 15 min. Detecting the levels of IL-1 beta, IL-6 and TNF-alpha in the serum of the mice by using an ELISA detection kit<0.001。
The above experimental results are consistent with those of FIG. 1A, BPOZ-/-And BPOZ+/-The level of IL-1 beta in the serum of the mice is obviously higher than that of BPOZ+/+Mouse (see B in fig. 1). In contrast, IL-6 (see FIG. 1C) and TNF- α (see FIG. 1D) which are dependent on Toll-like receptor signaling pathways are not significantly altered. The results of this study indicate that BPOZ depletion renders mice more susceptible to LPS-induced sepsis and affects the level of IL-1 β secretion that is dependent on the NLRP3 receptor pathway.
(II) construction of BPOZ+/+、BPOZ-/-Colitis model in mice, analysis of changes in sensitivity to inflammation including mouse body weight and mice before and after BPOZ deletionViscosity of rat feces, condition of feces blood, colon length observation, ulcer area displayed by HE staining, semi-quantitative evaluation of inflammation degree and the like.
The specific method and test results are as follows:
with BPOZ+/+、BPOZ-/-And BPOZ+/-The mice of the three genotypes are taken as research objects, 3% DSS (dextran sulfate sodium for inducing ulcerative colitis of the mice) is added into drinking water for 5 days continuously, and the death rate of the mice is calculated by observing the mice for 9 days continuously after drug withdrawal; adding 3% DSS into drinking water for 5 days, drinking common water for 15 days, and recording daily weight change, feces viscosity and hemorrhage; the mice were sacrificed after 5, 9 and 20 days, and colons were taken to observe their morphological changes, HE staining to observe their microscopic changes, colon length comparison, ulcer area comparison, etc.
See FIGS. 2(A) - (C) for the wild type (BPOZ)+/+N ═ 8) and BPOZ deficiency (BPOZ)-/-N-8) mice were fed drinking water containing 2% DSS for 5 days, followed by normal drinking water until the end of the experiment, and the mice were tested daily for body weight (see fig. 2 a), fecal consistency (see fig. 2B), and rectal hematochezia (see fig. 2C) after the start of 2% DSS feeding.
The above experimental results show that the body weight reduction of mice after BPOZ deletion is more significant compared to wild type (a of fig. 2); the results of scoring for stool viscosity and blood stool status show that the viscosity of the mouse feces is lower (B in fig. 2), the blood stool is more severe (C in fig. 2) and more difficult to recover after BPOZ loss.
Referring to fig. 2(D) - (E), on day 10, mice were taken for cecum, colon, rectum and spleen, colon length (D of fig. 2) was compared and photographed (E of fig. 2). The experimental results show that BPOZ-/-The colon of the mice is shorter (D-E in FIG. 2), indicating that it is severely compromised.
Referring to fig. 2(F) - (G), colons were subjected to HE staining (G of fig. 2) and semi-quantitative histopathological scoring (F of fig. 2) on day 5, day 10, and day 15 of the mice, respectively. The results show that BPOZ+/+The colon of the mouse had only a few ulcers (HE staining of F in fig. 2 shows the area of inflammation); the results of the semi-quantitative scoring also indicate BPOZ-/-Colon of mouseInflammation in comparison with BPOZ+/+The mice were more severe (G of fig. 2).
The above results all show that: BPOZ has a protective effect on DSS-induced colitis.
(III) culturing BPOZ+/+And BPOZ-/-Primary macrophages (BMDMs) of mice and human THP-1 cells with knocked-down BPOZ were analyzed for IL-1 β secretion by classical and non-classical stimuli such as NLRP3 stimuli (ATP, MSU, CPPD, Nigericin and Poly (I: C)). The specific method and test results are as follows:
(1) from BPOZ+/+And BPOZ-/-Isolation and culture of Primary macrophages (BMDMs) in mice, analysis of cells for IL-1 beta secretion with classical and non-classical stimuli
From BPOZ+/+And BPOZ-/-Primary macrophages (BMDMs) were isolated and cultured in mice and the level of IL-1. beta. in the cell supernatant was measured after treatment of the cells with NLRP3 stimuli (ATP, MSU, CPPD, Nigericin and Poly (I: C)), NLRP4 stimuli Flagellin, AIM2 stimuli PolydA: dT, non-classical signaling pathway stimuli (V.Cholerae, E.Coli and CTB).
Referring to FIGS. 3(A) - (C), the following are shown: from BPOZ+/+、BPOZ-/-Primary macrophages (BMDMs) were isolated and cultured in mice and the comparison of IL-1. beta. levels in the cell supernatants was examined by ELISA after treatment of the cells with NLRP3 stimulators ATP (5mM, 4h), MSU (200. mu.g/mL, 6h), CPPD (100. mu.g/mL, 2h), Nigericin (15. mu.M, 45min) and Poly (I: C) (2. mu.g/mL, 6h) (A in FIG. 3), NLRP4 stimulators Flagellin (1. mu.g/mL, 2h) and AIM2 stimulators PolydA: dT (2. mu.g/mL, 6h) (B in FIG. 3) and the non-classical stimulators V.cholere, E.coli, CTB (C in FIG. 3).
The above experimental results show that: under the action of these NLRP3 stimulators, mouse primary macrophages (BMDMs) enhance mature secretion of IL-1. beta. due to BPOZ deletion (see A of FIG. 3). In addition, Flagellin, a stimulator of NLRC4, polydA: dT, a stimulator of AIM2 (B in FIG. 3), and non-classical signaling pathway stimulators (V.cholere, E.coli and CTB) (C in FIG. 3) all also promote BPOZ-/-BMDMs (primary macrophage of BPOZ-deleted homozygous miceCells) secrete more IL-1 β. Experiments have shown that the deletion of BPOZ in BMDMs promotes IL-1 beta secretion mediated by various classical and non-classical inflammasome. This suggests that BPOZ has a negative regulatory role in classical and non-classical inflammatory body activation.
(2) Analysis of the secretion of IL-1 beta by BPOZ-knocked human THP-1 cells under classical and non-classical stimuli
Referring to fig. 3 (D): BPOZ-specific siRNA oligos (a small segment of RNA sequence can specifically inhibit BPOZ gene expression) are synthesized, a THP-1 cell is transfected for 24 hours by using Lipofectamine 2000 (a multifunctional transfection reagent) and is used for knocking down BPOZ (a sequence shown as SEQ ID NO: 2) in a human-derived THP-1 cell, cell lysate is collected, a BPOZ antibody is used for detecting the knocking-down efficiency of the BPOZ, and alpha-Tubulin (alpha Tubulin) is used as a control with equal load.
Human THP-1 cells with reduced BPOZ were treated with NLRP3 stimulator (ATP, MSU, CPPD, Nigericin), AIM2 stimulator polydA: dT and non-classical stimulator (CTB) and the secretion level of IL-1. beta. in the cell supernatant was varied by ELISA. As a result, as shown in FIG. 3(E), the level of IL-1. beta. was also increased in the supernatant of BPOZ-knocked-down cells.
The above results all indicate that BPOZ deletion in human THP-1 cells promotes IL-1 beta secretion mediated by classical and non-classical inflammatory bodies. This suggests that BPOZ has a negative regulatory role in classical and non-classical inflammatory body activation.
According to the above (1) to (2), it was confirmed that deletion or overexpression of BPOZ in both mouse and human cells may lead to excessive inflammatory diseases.
(IV) pro-caspase-1 and BPOZ with GFP labels are over-expressed in HEK293T cells, the interaction between the BPOZ and caspase-1 and active subunits caspase-1p20 and caspase-1p10 thereof is analyzed by a co-immunoprecipitation method, and the connection mechanism of the BPOZ and an inflammation signal path is further analyzed and determined. The specific method and test results include the following:
(1) co-immunoprecipitation analysis of whether BPOZ interacts with caspase-1 active subunit caspase-1p10/p20
HEK293T cells were used as the study subjects, GFP-pro-Caspase-1 (GFP-tagged pro-Caspase-1 plasmid), Flag-BPOZ and Flag empty vector were co-transfected into the cells, and the interaction of BPOZ with pro-Caspase-1 and the cleavant was detected by co-immunoprecipitation and immunoblotting. The results are shown in FIG. 4 (A). The experimental results show that: BPOZ interacts with caspase-1p10, indicating that BPOZ interacts with the caspase-1 active subunit.
In order to further clarify the interaction relationship, expression vectors of Flag-tagged caspase-1p10 and caspase-1p20 were constructed again, HEK293T cells were used as the study objects, HA-BPOZ, Flag-pro-caspase-1, Flag-caspase-1p 10, Flag-caspase-1p20 and Flag empty vectors were co-transfected into the cells, MG132 (protelomer inhibitor) was used to treat the cells for 8h, and the interactions of BPOZ with pro-caspase-1, caspase-1p10 and caspase-1p20 were detected by immunoprecipitation and immunoblotting. The results are shown in FIG. 4 (B).
The results of co-immunoprecipitation showed that the active subunits of both caspase-1, caspase-1p20 and caspase-1p10, interacted with BPOZ.
(2) Analysis of whether BPOZ-Cullin3 could associate with caspase-1p10/p20 subunit to form complex and affect stability of caspase-1p10, p20
HEK293T cells were used as the study subjects, Myc-Cullin1, Cullin2, Cullin3, Cullin4A, Cullin4B and Cullin5 were transfected into the cells, respectively, and Flag-BPOZ/Flag empty vectors (12 groups in total) were co-transformed, and the interaction between BPOZ and CULs was detected by co-immunoprecipitation and immunoblotting. As shown in FIG. 5(A), BPOZ interacts with Cullin3, which is consistent with the reported results, and it was also found that BPOZ also interacts with Cullin 4A.
In order to confirm the function of Cullin3 or Cullin4A in the process of BPOZ interaction with caspase-1 active subunit, by using dominant negative effect principle and taking HEK293T cell as a research object, respectively, the Dominant Negative (DN) mutant plasmids Flag-DN-Cullin3/Flag-DN-Cullin4A of Cullin3 and Cullin4A are co-transferred into HEK293T cells (group 2) together with HA-BPOZ, V5-caspase-1 p20 and Myc-Ub, and the influence of DN-Cullin3 and DN-Cullin4A on the stability of caspase-1p20 is detected by immunoblotting.
The results are shown in FIG. 5 (B): it was found that when Cullin3 was inactivated, the level of ubiquitination of caspase-1p20 was reduced or even eliminated; in contrast, caspase-1p20 still has ubiquitination when Cullin4A is inactivated. Thus, BPOZ was shown to affect the level of ubiquitination of caspase-1p20 by interacting with Cullin 3.
The HEK293T cells are taken as research objects, Flag-DN-Cullin3/Flag-DN-Cullin4A, HA-BPOZ, V5-caspase-1 p20 and Myc-Ub (totally 2 groups) are respectively transfected into the cells, and the influence of DN-Cullin3 and DN-Cullin4A on the ubiquitination level of caspase-1p20 is detected by using ubiquitination experiments and immunoblotting.
As a result, as shown in FIG. 5(C), it was found that when BPOZ was over-converted, the stability of caspase-1p20 was affected and the expression level was decreased; when Cullin3 was inactivated, the stability of caspase-1p20 was not affected and the change in expression level was not significant. In contrast, when Cullin4A was inactivated, the stability of caspase-1p20 was still affected and the expression level was still decreased (C in FIG. 5).
The above results demonstrate that the effect of BPOZ on the stability of caspase-1p20 is directly related to whether Cullin3 is inactivated, and not to Cullin 4A. The BPOZ is an adaptor protein of E3 ubiquitin ligase scaffold protein Cullin3, and plays a role of E3 ubiquitin ligase together with Cullin3 to influence the ubiquitination modification stability of caspase-1p 20.
And finally, taking HEK293T cells as research objects, co-transfecting HA-BPOZ, Myc-Cullin3 and Flag-caspase-1p 10/Flag-caspase-1p20 or Flag empty vectors (3 groups in total, Flag empty vectors are used as controls) into the cells, detecting HA-BPOZ, Myc-Cullin3 and Flag-caspase-1p 10/Flag-caspase-1p20 by immunoblotting after two-step co-immunoprecipitation, and confirming whether a complex can be formed among the HA-BPOZ, Culllin3 and caspase-1p10 (or caspase-1p 20).
The results of the experiment are shown in FIGS. 5(D) to 5 (E): BPOZ could be detected in both the cell immunoprecipitate complexes (precipitates) of caspase-1p20 (D in FIG. 5) and caspase-1p10 (E in FIG. 5). As described above, intracellular BPOZ can associate with Culllin3, caspase-1p10 (or caspase-1p 20) as a complex. Because the proteins of the CULs family (including Cullin3) interact with the protein containing the BTB structural domain, and the BTB structural domain can mediate ubiquitination of the target protein by the CULs as an adapter protein, when BPOZ is combined with Culllin3 and caspase-1p10 (or caspase-1p 20) to form a complex, the ubiquitination modification mode influences the stability of the caspase-1 active subunit (caspase-1 p10/p 20).
(3) BPOZ was determined to decrease maturation and secretion of IL-1 β by decreasing the stability of caspase-1 active subunits caspase-1p10, caspase-1p20
The effect of BPOZ on the stability of pro-caspase-1, caspase-1p10 and caspase-1p20 was examined by immunoblotting for quantification of Flag-pro-caspase-1, Flag-caspase-1p 10, Flag-caspase-1p20 and different doses (0, 0.3. mu.g, 0.6. mu.g, 0.9. mu.g) of Flag-BPOZ (total 12 groups) by transfection into HEK293T cells.
The results are shown in FIG. 6(A), and the experiment shows that BPOZ affects the stability of caspase-1p10 and caspase-1p20, but has no effect on the stability of pro-caspase-1.
Similarly, HEK293T cells were used as the study subjects, Flag-pro-caspase-1 and pro-IL-1. beta. were co-transfected into the cells, HA-BPOZ and HA empty vector were co-transferred, respectively, the effect of BPOZ on the stability of pro-caspase-1, pro-IL-1. beta., caspase-1p10, caspase-1p20 and IL-1. beta. p17 was examined by immunoblotting, and the level of IL-1. beta. secretion in the cell supernatant was examined by ELISA.
The experimental result is shown in FIG. 6(B), by reconstructing the mature release of signal molecule IL-1 beta downstream of NLRP3 inflammatory pathway in HEK293T cell, the result of immunoblot detection shows that when BPOZ is transgressed, the expression of pro-caspase-1 is not influenced, and the expression of caspase-1p10 and caspase-1p20 is obviously reduced; the expression of pro-IL-1 beta is not affected, and the expression level of mature IL-1 beta-p 17 is reduced obviously. As shown in FIG. 6(C), the secretion of mature IL-1. beta. in the cell supernatant was measured by ELISA, and it was found that the amount of IL-1. beta. in the supernatant was decreased by the BPOZ treatment.
The above results further demonstrate that BPOZ promotes the degradation of caspase-1 active subunits by targeting the constituent caspase-1 active subunits caspase-1p10 or caspase-1p20, thereby inhibiting the cleavage of IL-1 β precursor, and further inhibiting the mature secretion of IL-1 β.
When BPOZ was stimulated with NLRP3 stimuli (MSU, CPPD, Nigericin and Poly (I: C)), NLRP4 stimuli Flagellin, AIM2 stimuli PolydA: dT, respectively+/+And BPOZ-/-BMDMs cells (or LPS + ATP treated for different periods) were treated by immunoblotting to detect the level of caspase-1 cleavage in the cells.
The results are shown in FIG. 6(D), consistent with the previous conclusions, when BPOZ was treated with the NLRP3 stimuli MSU, CPPD, Nigericin and Poly (I: C), the NLRP4 stimuli Flagellin, AIM2 stimuli PolydA: dT+/+And BPOZ-/-BPOZ in mouse-derived BMDMs cells-/-The level of caspase-1p10 in the cells is obviously increased, and the mature secretion of IL-1 beta is increased. The same results were obtained by treating the cells with LPS + ATP for different periods of time (see E in FIG. 6).
The experimental conclusion shows that BPOZ can reduce the maturation and secretion of IL-1 beta by reducing the stability of caspase-1 active subunit caspase-1p10 or caspase-1p 20.
And (V) constructing a colitis lethal model of the mice and analyzing the sensitivity influence of the BPOZ overexpression mice on inflammation. The specific method and test results are as follows:
(1) construction of pAAV-BPOZ:
mouse intestinal over-expression BPOZ experiment, type 5 adeno-associated virus (AAV5) was selected, and pAAV-BPOZ virus packaging was entrusted to Henan bioscience (Shanghai) Co., Ltd.
The construction steps comprise:
firstly, plasmid construction: the mouse BPOZ gene was constructed into a type 5 AAV expression plasmid (pHBAAV-CMV-MCS-T2A-ZsGreen).
Packaging plasmids: 10. mu.g of the expression plasmid pHBAAV-BPOZ, 20. mu.g of the packaging vector pHelper, 10. mu.g of pAAV-RC were transfected into 293 cells.
③ AAV collection and purification: after 72 hours of transfection, all cells were collected and frozen and thawed repeatedly with liquid nitrogen three times to collect supernatant, which was then digested with Benonase enzyme, the resulting supernatant was purified with a purification column, and the resulting virus was stored at-80 ℃ after being split-packed.
Determination of AAV titer: the AAV is absolutely quantified by a fluorescent quantitative PCR method.
(2) Mice were fasted overnight and pretreated with 20mM N-acetyl-1-cysteine (NAC), and the colon was washed by intrarectal injection of 100 μ L20 mM NAC and allowed to dry for 15 minutes, repeating this procedure twice. Next, the mice were re-anesthetized and injected with 5X 1010 pAAV-null and pAAV-BPOZ via enema and tail vein, respectively, once a week for 3 weeks.
(3) Mice injected with pAAV-null or pAAV-BPOZ, respectively, were fed with 4% DSS (sodium dextran sulfate, 4% concentration induces ulcerative colitis and death in mice) for 7 consecutive days in their drinking water and normal drinking water after withdrawal until the end of the experiment, and mice mortality was observed and counted within 15 days.
The statistical results are shown in FIG. 7: the mortality rate of the mice injected with pAAV-BPOZ was significantly lower than that of the pAAV-empty mice. After 12 days of withdrawal, the mice injected with pAAV-null had all died, and the mice injected with pAAV-BPOZ had less than 50% mortality. The above results all show that: over-expression of BPOZ has an inhibitory effect on DSS-induced colitis.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> military medical research institute of military science institute of people's liberation force of China
Application of <120> BPOZ gene in preparation of medicine for treating excessive inflammatory response diseases
<141> 2021-02-04
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